C. P. W. Zebitz

Preparation and purification of DNA from insects for AFLP analysis

Analysis of amplified fragment length polymorphism (AFLP) has the potential to become a powerful new DNA fingerprinting technique for studying genetic relationships and genetic diversity in arthropods. Since DNA of high quality is a crucial prerequisite for AFLP analysis we evaluated the applicability of six protocols (one fast and four complex methods with phenol‐chloroform treatments as well as one CTAB‐based method) for extracting DNA from insect material and three additional DNA purification steps. The most rapid DNA isolation method did not produce DNA suitable for AFLP analysis. Among four complex methods tested, two protocols resulted in comparatively low yields of DNA that was therefore not used as template for AFLP analysis. The other two complex methods with phenol treatments and a CTAB‐based DNA extraction protocol provided DNA suitable for AFLP assay. An additional purification of the DNA using spermine precipitation revealed a few extra bands in an AFLP gel that were masked in unpurified DNA. Therefore spermine precipitation is recommended for AFLP templates.

Rapid emergence of baculovirus resistance in codling moth due to dominant, sex-linked inheritance.

Insect-specific baculoviruses are increasingly used as biological control agents of lepidopteran pests in agriculture and forestry, and they have been previously regarded as robust to resistance development by the insects. However, in more than a dozen cases of field resistance of the codling moth Cydia pomonella to commercially applied C. pomonella granulovirus (CpGV) in German orchards, resistance ratios exceed 1000. The rapid emergence of resistance is facilitated by sex-linkage and concentration-dependent dominance of the major resistance gene and genetic uniformity of the virus. When the gene is fixed, resistance levels approach 100,000-fold. Our findings highlight the need for development of resistance management strategies for baculoviruses.

Effect of some crude and azadirachtin-enriched neem (Azadirachta indica) seed kernel extracts on larvae of Aedes aegypti

Bioassays against larvae of Aedes aegypti were conducted with neem seed kernel extracts obtained by extraction with water and organic solvents. Permanent exposure of fourth instar larvae to treated water resulted in a conspicuous growth‐disrupting effect, mainly during imaginai development. The effectiveness of the extracts increased with decreasing polarity of the solvents used for extraction. Three neem seed kernel extracts caused an extreme prolongation of the larval period when first instar larvae were continuously exposed to treated water until adult emergence. The time necessary for lethal action of neem seed kernel extracts to set in was similar to that reported for some synthetic IGRs.

Physical and chemical plant characters inhibiting the searching behaviour of trichogramma chilonis

Several plant characters are known to affect the searching behaviour and parasitization efficiency of Trichogramma spp. (Hymenoptera: Trichogrammatidae). In this study, plant characters contributing to the low Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) egg parasitism levels on pigeonpea (Cajanus cajan (L.) Millspaugh) were investigated. The efficiency of T. chilonis on pigeonpea was dependent on the plant structure on which the host eggs were found. In a cage experiment, more than 55% of eggs placed on leaves were parasitized, while 1% of eggs on calyxes and no eggs on pods were parasitized. In a filter paper bioassay, parasitoids were deterred by acetone and hexane surface extracts from pigeonpea pods but showed no response to water extract. The searching behaviour of the parasitoids was not affected by different solvent extracts from the surface of pigeonpea leaves. In a four‐armed airflow olfactometer, T. chilonis was repelled by volatiles from pigeonpea pods but showed no response to volatiles derived from hexane extract of pod surfaces. Volatile infochemicals and hexane surface extracts from pods of two wild Cajanus species, C. scarabaeoides (L.) Thours and C. platycarpus (Bentham) van der Maesen, were similarly deterrent to T. chilonis. The movement of the parasitoids on pigeonpea pods and calyxes was inhibited by long trichomes and wasps were trapped by sticky trichome exudates. Parasitoids walked significantly faster on leaves than on pods. The walking speed on both pods and leaves increased significantly after washing with hexane. The results presented in this paper show that the plant growth stage and the plant structures preferred by H. armigera for oviposition are the least suitable for T. chilonis, contributing to the low parasitoid efficiency on pigeonpea.

Molecular interactions between rosy apple aphids, Dysaphis plantaginea, and resistant and susceptible cultivars of its primary host Malus domestica

The use of crop varieties resistant or tolerant to insect pests or other stress factors is one approach in non‐chemical crop‐protection. Knowledge of the biochemical and molecular background of insect–plant interactions is a prerequisite for optimizing breeding for resistance. However, the resistance genes involved in plant–aphid interactions have so far only been identified and characterized in very few plant species. Our work aims to elucidate the molecular and biochemical mechanisms involved in resistance of apple trees, Malus domestica L. (Rosaceae), against its primary aphid pest, the rosy apple aphid, Dysaphis plantaginea (Passerini) (Homoptera: Aphididae), which is considered a serious economic pest of apple. Gene expression in both resistant and susceptible apple cultivars after infestation with rosy apple aphids was investigated by employing the cDNA‐AFLP method (cDNA–Amplified Fragment Length Polymorphism). From approximately 12 500 cDNA fragments detected on polyacrylamide gels, 21 bands were apparently up‐ or down‐regulated only in the resistant cultivar ‘Florina’ after aphid infestation compared to the susceptible cultivar ‘Topaz’ and/or mechanically wounded or non‐infested leaves. These fragments were cloned, sequenced, and the pattern of gene expression for six fragments was subsequently verified by virtual Northern blots. Sequence comparisons of these fragments to GenBank accessions revealed homologies to already known genes, most of them isolated from Arabidopsis thaliana L. Among them, a putative RNase‐L‐inhibitor‐like protein, a pectinacetylesterase, an inositol‐phosphatase‐like protein, a precursor of the large chain of the ribulose‐1,5‐biphosphate‐carboxylase, and defence‐related genes such as a vacuolar H(+)‐ATPase subunit‐like protein and an ADP‐ribosylating enzyme were identified. The results are discussed in relation to a putative role of these genes in conferring aphid resistance in apple trees.

Amplified fragment length polymorphism analysis of different geographic populations of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae)

The gypsy moth, Lymantria dispar Linnaeus, is one of the most serious insect pests of palaearctic and nearctic hardwood forests. We used amplified fragment length polymorphism (AFLP) to detect genetic diversity within and among gypsy moth populations. Five AFLP primer combinations were used on 98 L. dispar samples from different parts of Europe, Asia and North America, detecting a total of 481 polymorphic and 58 monomorphic fragments. Genetic similarities based on these data were calculated and cluster analysis was performed to graphically display groupings between isolates. Lymantria dispar individuals from close geographical areas of Europe were mostly grouped together in cluster analysis resulting in the formation of subgroups corresponding to the origin of the samples. Supporting this observation, clustering of individuals from 22 neighbouring populations in southern Germany agreed well with the region they originated from. Thus, AFLP analysis revealed the existence of a certain degree of genetic variability between European gypsy moth populations that could be explained by the accumulation of polymorphisms resulting from both historical population bottlenecks and the adaptation to different environmental conditions. The results of this study therefore demonstrate that AFLP analysis is a sensitive technique for distinguishing genotypes from different geographic origins as well as from neighbouring local populations and provides sufficient molecular markers for future characterization of the gypsy moth genome.

On the overwintering ability of Drosophila suzukii in South Tyrol.

BACKGROUND: Drosophila suzukii became one of the most dangerous pests in fruit crops and vineyards in Europe since 2009. Lacking knowledge of the overwintering biology under European environmental conditions makes an environmentally friendly control of this invasive pest difficult. OBJECTIVE: The objective of this study was to investigate its hibernation capability under the climatic conditions of South Tyrol, Italy. METHODS: Monitoring flight activity by baited traps revealed an overwintering location in the Adige valley. We determined the reproductive status of females trapped weekly at “Schlossleiten” from August 2012 until September 2013 by dissecting their abdomens. For further potential correlation between overwintering locations, reproductive status, and climatic conditions were assessed. RESULTS: The microclimatic conditions of single locations affected overwintering success and suggest the aggregation of adults in most favourable hibernation sites where a high proportion of females is able to survive. However, in spring some of the females showed degenerated reproductive organs. Dissection of abdomens evinced a reproductive diapause, which appears to be affected by winter climatic conditions, the nutritional status, and food availability. CONCLUSIONS: D. suzukii is able to overwinter in the South Tyrolean area. Control and management of the potential hibernation sites may delay the population dynamics in the following season.

Effects of three different neem seed kernel extracts and azadirachtin on larvae of different mosquito species

The insect growth‐regulating effects of neem seed kernel extracts and crude and pure azadirachtin on mosquito larvae were investigated in the laboratory and compared with the effects of the juvenile hormone analogue Altosid. The susceptibility to neem seed kernel extracts, in decreasing order, of young fourth instar larvae of the species tested was Aedes togoi (Theobald) δ Anopheles stephensi List., Culex quinquefasciatus Say, Aedes aegypti L. Azadirachtin as well as seed kernel extracts exerted a pronounced growth‐regulating effect during imaginal development. Differences between azadirachtin or seed kernel extracts and Altosid could be pointed out considering the steepness of slope of the ldp‐lines and the susceptibility of fourth instar larvae of different age classes to either the JHA or the natural product.

Tarsal morphology and attachment ability of the codling moth Cydia pomonella L. (Lepidoptera, Tortricidae) to smooth surfaces

Despite several studies on the attachment ability of different insect taxa, little is known about this phenomenon in adult Lepidoptera. In this study we combined morphological and experimental analyses of tarsal adhesive devices and the attachment ability of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) to smooth surfaces. Pretarsi of C. pomonella attach to smooth substrates by means of their smooth, flexible and well developed arolia. Using the centrifugal force measurement technique, friction forces of males and females were assessed on hydrophobic and hydrophilic glass surfaces. Adults of both sexes generated similar forces in spite of the noticeable difference in their body masses. That is why males showed significantly higher safety factors (attachment force divided by body weight) compared to those of females. Hydrophobicity of the substrate had no considerable effect on friction forces. For females, friction forces (sliding parallel to the substrate plane) were compared with adhesive forces (pulling off perpendicularly from the substrate plane) measured on Plexiglas surfaces. It can be concluded that the attachment system of C. pomonella is rather robust against physico-chemical properties of the substrate and is able to achieve a very good attachment on vertical and horizontal substrata.

Response of Trichogramma egg parasitoids to colored sticky traps

The response of Trichogramma spp. egg parasitoids to colored sticky traps was evaluated in the field during two seasons (1995/1996, 1996/1997). Traps consisted of a glass tube coated with Bird-Tanglefoot® into which colored paper was inserted or clear traps without paper. Colors tested were white, green, blue, yellow and red in the first season and white, green, yellow and black in the second season. The proportion of both female and male parasitoids caught on the sticky traps was significantly different among colors, indicating that the parasitoids actively move between plants and are not solely carried along passively by wind. White was the color most preferred by female parasitoids, followed by clear and green traps. Yellow was preferred over black but was less attractive than green. Visual cues may be used by Trichogramma spp. during the habitat location process. The color preference of male Trichogramma spp. differed significantly from females with yellow and green being more attractive than white. For all colors, more female Trichogramma spp. were caught on the sticky traps (>85% of all wasps caught), indicating a lower activity level and/or shorter lifespan for males. The use of white cylindrical sticky traps for monitoring Trichogramma spp. populations in the field is recommended.